The role of Pseudomonas aeruginosa ExoS during early pneumonia.

描述(申请人提供)：铜绿假单胞菌是一种革兰氏阴性病原体，是医院获得性肺炎的主要原因。为了引起严重感染，铜绿假单胞菌使用III型分泌系统将效应蛋白直接注入真核细胞的细胞质。目前发现的四种效应蛋白是ExoU、ExOS、ExoT和ExoY。在人类和动物的急性肺炎模型中，ExoU和EXOS与严重疾病的联系最为密切。有趣的是，大多数临床分离株只分泌ExoU或EXOS，而不是两者都分泌，这表明它们在致病过程中起着相似或多余的作用。了解这两种酶性质不同的毒素如何使铜绿假单胞菌引起严重肺炎，将为开发预防和治疗铜绿假单胞菌感染的疗法奠定基础。本项目旨在了解EXOS在肺炎中的致病机制。初步数据显示，分泌exos的菌株大量存在于肺部，而该基因的缺失会导致在感染的最初24小时内迅速从肺部清除。EXOS+菌株能够持续存在，尽管诱导中性粒细胞和巨噬细胞强劲地募集到肺中，这表明EXOS以某种方式使这些细胞丧失能力。该项目将确定EXOS是否通过在肺炎早期损害吞噬细胞来阻止铜绿假单胞菌从肺中清除。使用急性肺炎的小鼠模型和优化的基于FRET的报告分析，将识别在急性肺炎早期阶段注射EXOS的细胞类型。在肺炎早期，EXOS中毒对吞噬细胞的影响将通过检测吞噬细胞对细菌的内化、这些细胞对细菌的杀灭和吞噬细胞的存活来确定。各种体外和体内试验，包括基于荧光的内化试验，将被用来研究这种对吞噬细胞的影响。这些目标将确定EXOS的细胞靶点，以及在肺炎早期，EXOS中毒对这些细胞的影响。这些信息与目前关于ExoU机制的知识相结合，将有助于更好地了解感染期间铜绿假单胞菌的整体发病机制。 公共卫生相关性：铜绿假单胞菌是一种重要的医院病原体，每年在美国造成约64万人感染。由于这些感染的严重性和频率，需要确定铜绿假单胞菌的致病机制，以更好地预防和治疗铜绿假单胞菌感染。这项应用的目标是更好地了解这种医学上重要细菌的发病机制。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is a gram-negative pathogen that is the major cause of hospital-acquired pneumonia. To cause serious infections, P. aeruginosa uses a type III secretion system to directly inject effector proteins into the cytoplasm of eukaryotic cells. The four effector proteins currently identified are ExoU, ExoS, ExoT, and ExoY. ExoU and ExoS have been most closely linked to severe disease in human and animal models of acute pneumonia. Interestingly, most clinical isolates secrete either ExoU or ExoS but not both, suggesting that they play functionally similar or redundant roles in pathogenesis. Understanding how these two enzymatically different toxins both allow P. aeruginosa to cause severe pneumonia will lay the foundation to develop therapeutics to prevent and treat P. aeruginosa infections. This project aims to understand the pathogenic mechanism of ExoS in pneumonia. Preliminary data show that ExoS- secreting strains persist in the lungs at high numbers whereas deletion of the gene results in rapid clearance from the lung over the initial 24 hr of infection. ExoS+ strains are able to persist despite eliciting robust recruitment of neutrophils and macrophages to the lung, suggesting that ExoS somehow incapacitates these cells. This project will determine if ExoS acts to prevent P. aeruginosa clearance from the lungs by impairing phagocytes during early pneumonia. Using a mouse model of acute pneumonia and an optimized FRET-based reporter assay, the cell types injected with ExoS during the early stages of acute pneumonia will be identified. The effect of ExoS intoxication on phagocytes during early pneumonia will be defined by examining bacterial internalization by phagocytes, killing of bacteria by these cells, and phagocyte survival. Various in vitro and in vivo assays, including a fluorescence-based internalization assay, will be used to study this effect on phagocytes. These aims will define the cellular targets of ExoS and the consequences of intoxication with ExoS on these cells during early pneumonia. Such information, in conjunction with the current knowledge on the mechanisms of ExoU, will lead to a better understanding of the overall pathogenesis of P. aeruginosa during infection. PUBLIC HEALTH RELEVANCE: P. aeruginosa is an important nosocomial pathogen causing approximately 640,000 infections per year in the United States. Due to the severity and frequency of these infections, the pathogenic mechanisms of P. aeruginosa need to be determined to better prevent and treat P. aeruginosa infections. The goal of this application is to better understand the pathogenesis of this medically important bacterium.